Effect of enzymatic modification on the structure and rheological properties of diluted alkali soluble pectin fraction rich in RG-I

doi:10.21203/rs.3.rs-3787062/v1

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Effect of enzymatic modification on the structure and rheological properties of diluted alkali soluble pectin fraction rich in RG-I

https://doi.org/10.21203/rs.3.rs-3787062/v1

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This study focuses on pectin covalently linked in cell walls and extracted using diluted alkali (DASP) from two sources: apples and carrots, describing changes in rheological properties due to enzymatic treatment. Given DASP's richness in rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on FTIR spectra. ABF treatment led to decrease in stability of pectic gels while simultaneous use of ABF, RGAE and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, involving side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Biological sciences/Biochemistry/Carbohydrates

Biological sciences/Biochemistry/Enzymes

Biological sciences/Plant sciences/Plant cell biology/Cell wall

Physical sciences/Chemistry/Chemical biology/Carbohydrates/Dietary carbohydrates

Physical sciences/Chemistry/Chemical biology/Carbohydrates/Polysaccharides

Pectin constitute up to 35% of plant cell walls and perform there important functions in plant growth and development, maintain cell-cell integrity and, in the case of fruit and vegetables, determine the firmness and texture ^1,2. Pectin are considered as the component providing visco-plastic properties to the load bearing cellulose-hemicellulose network in cell wall, therefore play an important component for plant cell wall rheology ³. Pectin are important also for food industry due to its ability to increase viscosity and bind water. Moreover, pectin gelling activity may be tuned by various parameters, such as the type of pectin, concentration, pH, temperature, and the presence of cations ^4,5.

There are three main pectic domains. Homogalacturonan (HG) consists of a linear chain of α-(1, 4)-linked D-galacturonic acid (GalA) and is known as pectins “smooth” region. Rhamnogalacturonans belong to so called “hairy region”. The backbone of RG-I is made of the diglycosyl repeating unit [→4-α-d-GalpA-(1→2)-α-l-Rhap-(1→]. Predominantly, a large proportion of rhamnose units are substituted at O-4 with side chains composed principally of arabinans, galactans and/or arabinogalactans ¹. Rhamonogalacturonan II (RG-II) is a substituted galacturonan with α-(1, 4)-linked D-galacturonic acid backbone and four different glycosidic complexes as side chains.

Neutral side chain loss and the rearrangement of their associations within RG-I are one of the most pronounced and earliest changes in pectin structure during maturing, ripening and storage in fruit and vegetables. The majority of structural changes is associated with β-galactosidase and α-L-arabinofuranosidase enzymatic activity, observed during ripening, especially for firmly bound polymers, extracted by sodium carbonate ⁶. The percentage of methylesterified GalA units within the HG substructure is defined as the degree of methyl esterification (DM), while percentage of or O-acetylated GalA units is degree of acetylation (DA). The amount of methyl and acetyl groups in pectin chain affect the gelling conditions and the viscosity of pectins solutions, and thus it is one of the major factors in the determination of their functionality. Degree of acetylation and degree of methylation are strongly influenced by plant source as well as the extraction method. For low-methylated (LM) pectin (DM < 50%), gelation occurs at acidic pH (2–6) and presence of divalent ions such as Ca²⁺, while high-methylated (HM) pectin (DM > 50%) form gels in the presence of more than 55% sugar or a similar co-solute at pH < 3.5 ⁷. Hydrogen bonds and electrostatic interactions play a crucial role in the gelation mechanism of HM-pectin, while for LM-pectin the “egg-box” model describes binding processes and junction zones formation between non-esterified GalA units and calcium ions ⁸. Features of the polymer backbone of pectin such as its intrinsic flexibility or stiffness play major role in the rheological properties in solution and influence the order/disorder state of the system on a supermolecular scale, especially while different levels of chain association may be involved in network formation ⁹.

The diluted alkali-soluble fraction of the cell wall pectic matrix extracted with sodium carbonate (DASP) is considered as the fraction covalently linked in cell wall. It was demonstrated that these molecules shows distinctive features of creating a self-assembled network on mica ¹⁰. AFM imaging and coarse grain simulations confirmed that the network - like appearance on mica originates from rhamnose units separating two sections of homogalacturonan and creation of the kinks at characteristic angle of 118° ¹¹. Previous studies demonstrated that DASP pectin from fruit like pear or apple show concentration, pH or mono and divalent cations dependent gelling ability in aqueous medium, indicating possible application of this polysaccharide fraction due to their low methylation level ¹². However, due to significantly different conformation of RG-I than HG ² we hypothesize that rheology of DASP, that is rich in RG-I ¹³, may be largely affected by the side chains and the presence of the kinks caused by rhamnose interspacing galacturonic acid.

The goal of this paper is characterization of changes in the structure and resulting rheological properties of diluted alkali soluble pectin (DASP) fraction, extracted from two horticultural sources (apple and carrot), structurally changed by enzymatic modifications. In this experiment RGI acetyl esterase, rhamnogalacturonan endolyase and arabinofuranosidase are used. RGI acetyl esterase (RGAE) is an enzyme that participates in the deacetylation of D-GalA in the RGI backbone. Rhamnogalacturonan endolyase (RGL) participates in the endotype eliminative cleavage of L-α-rhamnopyranosyl-1,4-α-D-galactopyranosyluronic acid bonds of rhamnogalacturonan I domains in ramified hairy regions of pectin leaving L-rhamnopyranose at the reducing end and 4-deoxy-4,5-unsaturated D-galactopyranosyluronic acid at the non-reducing end. Arabinofuranosidase (ABF) preferentially removes α-1,2- and α-1,3-linked arabinose from side chains from either arabinan or arabinoxylan, and hydrolyses α-1,5-linked arabinooligosaccharide at low rate. In order to elucidate differences among pectin sources, the effect of selective modification of the pectin chain was compared for apple and carrot, and studied by HPLC, FTIR spectroscopy, AFM imaging with image analysis and confronted with rheological measurements of pectin aqueous solution.

Nanostructure

Representative AFM height images of DASP from apple and carrot in control (buffer) batch and after 120 min of incubation in three enzymatic cocktails (E1, E2 and E3) are shown in Fig. 1. Zoomed regions presented in Fig. 1a, f of the DASP incubated in the buffer only shows fibers in the form of rod-like structures with linear segments of variable length and separated by bending or branching points. Such features have been previously observed for DASP fraction extracted either from apple, carrot or pear ^10,14 and were explained as the result of rhamnose interspersion with galacturonic acid ¹¹. A similar structure on the mica was created by the DASP fibers after applying the E1 treatment (Fig. 1b, g). In the variant treated with E2 (Fig. 1c, h), larger aggregates and longer chains were observed, which was particularly pronounced for carrots (Fig. 1h). Additionally, both in the case of E2 treatment and the combination of three enzymes (E3) (Fig. 1d, i), short chains and small molecules were mostly noted.

Thanks to image analysis performed on the AFM scans, the observed structures were categorized into “hairy” molecules or “smooth” molecules. Additionally, total length of the branched molecule was calculated as the sum of lengths of branches belonging to molecule. Average total lengths of the molecules classified as “hairy”, before placing in buffer, were about 585 ± 23 nm and 443 ± 23 nm for apple and carrot DASP, respectively (Fig. 2a). This length is consistent with previously obtained by ¹⁵ that was reported to be from 20 to 1000 nm for the sodium carbonate pectin fraction extracted from different fruits. Figure 2a shows that placing pectin in buffer for 120 min (no enzymes yet) caused that the length of “hairy” molecules did not changed for apples, while increased for carrot. The length of molecules classified as “smooth”, before placing in buffer (Fig. 2b), was shorter than 100 nm and the incubation with buffer for 120 min caused apparent shortening in the case of apple while a slight increase in the case of carrot (Fig. 2b). The effect of incubation with buffer, reflected by changes in parameters after 120 min of incubation, suggests that pectins incubated in buffer alone may undergo structural changes leading rather to self-aggregation. Since pectin were incubated in buffer at pH 7, this result may be referred to previously obtained for DASP from apple and explained by the mechanism of high electrostatic repulsion between fully dissociated macromolecules that probably blocked the formation of extended pectin chains ⁴.

Enzymatic treatment with cocktails E1 and E3 did not cause statistically significant change of the length of the “hairy” molecules extracted from apple (Fig. 2a). In the case of carrot (Fig. 2a), treatment in E1 caused slight increase but, not significant, of the length, while treatment in E3 caused statistically significant decrease, when compared to incubation with buffer. From analysis of number of segments (Fig. 2d) and the average length of segments (Fig. 2c), one may notice that the total length of “hairy” molecules relates to the number of segments, but simultaneously the segments seems to become slightly shorter after incubation with enzymes. The most pronounced effect on the structure of DASP pectins in both materials has been obtained when E2 treatment was applied (Fig. 2a). Arabinofuranosidase (E2) that was exclusively active in the E2 caused significant increase of the total length of “hairy” molecules (up to almost 1.5 micrometer after 120 min). This effect was clearly associated with the increase of number of segments and their only slight shortening during incubation (Fig. 2d). It is also worth to note that the chains classified as “smooth” (Fig. 2b) had length similar to length of segments in the “hairy” molecules (Fig. 2b), i.e. less than 100 nm. The “smooth” molecules were unaffected by enzymatic treatments even with E2 (Fig. 2b). As ABF preferentially removes α-1,2- and α-1,3-linked arabinose from side chains from either arabinan or arabinoxylan, the effect of E2 on the structure of pectins molecules could be explained by the gradual removal of arabinose followed by aggregation of RG-I molecules resulting in three-fold increase in the number of branches per molecule (from about 5 to 15 segments per molecule) showed in Fig. 2d.

Contrary to ABF applied alone, the simultaneous action of ABF with RGAE and RGL enzymes (E3 treatment) did not result in aggregation and did not increase in length of “hairy” molecules. The length of “hairy” molecules after incubation in E3 was equal to 576 ± 26 nm and 356 ± 12 nm for apple and carrot DASP, respectively. A decrease in the number of branches per molecule, from 7 side branches for both sources, to 6 for apple and 5 for carrot pectins was also observed for this treatment. Shortening of “hairy” molecules and decrease in the number of branches due to E3 was more pronounced for carrot than for apple DASP. Probably this higher fragmentation of the carrot pectin chain may result from the greater RG-I content (62.90 mol%) than in apple (41.50 mol%), as it was previously described ¹³, providing more sites of action for pectinolytic enzymes.

Incubation in E1 enzyme mixture did not cause significant differences in branches length (Fig. 2c) for both sources. It is suspected that the function of the RGL enzyme may have been impaired due to the abundance of arabinose side chains preventing access to the chain as steric hindrance.

Slight decrease of segment length (Fig. 2c), that could be attributed to the length of side branches, was noted for E2 treatment. Moreover, for the combination of three enzymes (E3), a decrease in the average length of smooth molecules (Fig. 2b) was also observed indicating chains fragmentation. It may suggest that the simultaneous action of the enzymes modifying the RG-I skeleton and the enzymes that remove the arabinose side chains allowed for more effective fragmentation of the pectin chains. Hence, it supports the above explanation that the arabinose abundant in large amounts in the studied fraction could limit access of enzymes that modify the RGI backbone.

Functional groups

Since the intrinsic characteristics of the material determine its subsequent properties, Table 1 presents the acetyl and methyl content of the native initial pectin materials. Degree of acetylation (DA) was 5.59% and 7.48% for apple and carrot, respectively, while degree of methylation (DM) was not detected, suggesting the de-esterification of the pectins during sodium carbonate extraction ¹⁶.

Table 1

Degree of acetylation and degree of methylation of native DASP in apple and carrot. nd., not detected. The asterisks indicate statistically significant differences (t-Student test, p = 0.05).
	DASP source
	Apple	Carrot
DA (%)	5.59 ± 0.28*	7.48 ± 0.26*
DM (%)	nd.	nd.

The collected FTIR spectra for apple and carrot, native and treated with E3 for 120 min, are shown in Fig. 3. The overall shape of a polysaccharide spectrum is determined by the polysaccharide composition of backbone but can also be strongly influenced by the side chain constituents ¹⁷. The wavelength and intensity of the bands allow to evaluate possible changes in polysaccharide composition. For all samples, characteristic absorption regions can be distinguished. The shape of each spectrum had similar pattern, characteristic for DASP polysaccharides ^2,16,18. Region in the range of wavelength of 3600–3100 cm^− 1 is assigned to stretching vibration bands of hydroxyl groups and is associated with inter- and intramolecular hydrogen bonding. A higher intensity of this peak is observed for enzymatically modified pectins, which indicates a higher water absorption. Bands of C-H deformation vibrations appear at 3000–2800 cm^− 1. For all samples, absence of peak in the range of 1745–1700 cm^− 1 originates from vibration of esterified groups, confirmed lack of methyl esters in studied fraction of pectin, quantified by HPLC measurements.

Band around 1590 cm^− 1 is assigned to COO − antisymmetric stretching polygalacturonic acid, representing non-esterified carboxyl groups in pectins, while peak at about 1407 cm^− 1 represents symmetric carboxylic anions stretching vibrations. Bands at 1330 cm^− 1 (assigned to ring vibration) and 1240 cm^− 1 (C-O stretching vibrations in pectin), both characteristic to DASP fraction, were present for all samples. Based on FTIR spectra absorbencies in the range of 1200–900 cm^− 1 (fingerprint region) it is possible to determine groupings that are specific for each polysaccharide. The main component influencing the change of the shape of this fingerprint region due to enzymatic treatment is GalA, showing main absorbance regions at about 1140, 1090, 1070, and 1030 cm^− 1. However it has been shown, that also different pectic compounds can show different characteristic position of bands maximum in this region ^18,19. The absorbance bands at about 1075 cm^− 1 and 1045 cm^− 1 suggest also presence of RG-I domains ¹⁷. Changes in intensity of bands in this region and/or the disappearance of the peaks, observed for modified fractions, may suggest fragmentation changes of the DASP main chain. The peak at about 950 cm^− 1, characteristic of RG-I ¹⁸, assigned to galactose side chains ¹⁰, decreased in intensity for the E3 modified apple DASP, while its disappearance was observed for the enzymatically treated carrot fraction. This could indicate a degradation of galactan side chains, as the result of rhamnose removal. Bands in the wavenumbers in the range of 900–800 cm^− 1, belong to anomeric region, and can be used to differentiate the α and β configuration of anomeric carbon. Peaks at about 890–850 cm^− 1 indicate presence of galactopyranose and arabinofuranose units in sample ¹⁷. Greater changes in this region of spectra was observed for enzymatically modified samples from carrot.

Rheological properties

Flow and viscosity curves collected after 120 minutes of treated in buffer and in enzymes are showed in Fig. 4. Power law (Ostwald de Waele’s) and Herschel–Buckley’s flow models were fitted to the shear rate-shear stress curves for all samples. The consistency coefficient (K) and flow behaviour index (n) were used to described fluids behavior (Table 2). All n values were less than 1, showing that samples behave as pseudoplastic shear-thinning fluids, as reported previously for other pectin solutions ²⁰. This indicates that their apparent viscosity decreases with increasing shear rate and macromolecular network gets orientated or deformed in the direction of flow.

The E2 (ABF) treatment led to decrease in consistency coefficient value for both fractions, which indicates a weakening of the binding in the network. It was concluded that arabinose side chains were involved in macromolecular entanglements in native fraction, which resulted in higher viscosity for the pectins in buffer ²¹. The high impact of ABF on the structure of DASP could be caused by the relatively high content of arabinose in tested fraction. According to previous studies ¹³ the content of this monosaccharide was 24.9 ± 1.6 mol% in apple DASP and 18.2 ± 0.9 mol% in carrot DASP. Moreover, the tested fractions differed in rhamnose content – 3.8 ± 0.2 mol% for apple DASP and 8.4 ± 1.8 mol% for carrot DASP. It is worth noticing that for apple DASP, containing higher amounts of arabinose, decrease in the viscosity value and pseudoplastic character after incubation in ABF was much more pronounced. The suggested role of arabinose in the formation of a compact network was supported by the decrease in the value of the parameter G₀ (yield stress) for treated samples with ABF, describing the minimum shear rate needed to initiate flow of the material. On the other hand, high increase of this parameter, observed for E3 variant, indicated the formation of a dense network, resistant to mechanical disruption, for debranched polymer. Increase in K coefficient for E3 variant, combined with decrease in flow index, suggests stronger pseudoplastic character of DASP solution after simultaneous deacetylation and removal of Ara and Rha. The carrot DASP sample, after modification with this enzymatic cocktail, showed the highest pseudoplasticity of all tested solutions. The E3 resulted also in an increase of viscosity value for both sources. This may indicate a greater possibility of particles movement controlled by the entanglements of side chains attached to the rhamnose units as well as acetyl groups, which can hinder adoption by the polymer of binding-favorable conformations ²². In addition, rhamnose inclusions themselves can limit cross-linking of chains ²³. The increase of viscosity of DASP was previously observed during storage of carrot roots ²⁰. It was hypothesized there that hydrogen bonding between smooth pectin chains and hydrophobic interactions by the methyl groups of pectin chains have been formed, as a result of enzymatic modification naturally occured in roots. Therefore it is suspected that E3 treatment resulted in predominantly unbranched acid polymers. Taking into account low DM as result of extraction with sodium carbonate, under these conditions the tendency to self-aggregate in deionized water is reduced, hence molecules adopt a more extended conformation, favoring interactions between the chains ^24,25. This interpretation is supported by recently highlighted role of intermolecular interactions in the mechanical properties of LM pectins, which extended conformation at neutral pH increased the elastic character of the mixture ²⁶.

Table 2

Rheological properties of native and modified DASP from apple and carrot. G’, elastic (storage) modulus; G’’, viscous (loss) modulus; tanδ, loss factor; K, consistency coefficient; n, flow index; G₀, initial shear stress. Different letters indicate statistically significant differences (ANOVA, p = 0.05).
Parameter	Unit			Pectin source
				Apple				Carrot
				BUFFER	E1 RGAE + RGL	E2 ABF	E3 RGAE + RGL +ABF	BUFFER	E1 RGAE + RGL	E2 ABF	E3 RGAE + RGL + ABF
G'	Pa			310.83 ± 231.30^ab	1154.61 ± 139.91^a	32.29 ± 17.23^ab	338.77 ± 145.85^ab	405.53 ± 28.92^b	167.26 ± 154.87^ab	43.67 ± 25.38^ab	207.33 ± 119.49^ab
G'/G''				7.52 ± 3.14^ab	10.74 ± 1.42^b	4.38 ± 0.99^ab	8.34 ± 1.43^ab	6.77 ± 0.51^ab	6.44 ± 1.08^ab	3.51 ± 0.93a	6.93 ± 2.48^ab
tanδ				0.14 ± 0.00^abc	0.09 ± 0.01^bc	0.24 ± 0.05^a	0.13 ± 0.03a^b	0.15 ± 0.01^abc	0.16 ± 0.03^abc	0.30 ± 0.07c	0.15 ± 0.07^ab
Flow point	%			6.07 ± 3.83^ab	5.76 ± 1.26^bc	16.09 ± 4.50a^b	3.77 ± 1.95^a	1.93 ± 0.85^a	12.85 ± 7.76a^bc	20.60 ± 10.64c	3.77 ± 1.62^ab
Linear viscoelasticity limit	%			3.72 ± 1.83^abc	1.79 ± 0.50^bc	5.76 ± 2.20^a	1.54 ± 0.75^a	1.34 ± 0.90^a	3.41 ± 1.47^abc	7.20 ± 2.23c	1.88 ± 0.79^ab
Power law model	K	Pa s		2.59 ± 3.99^a	1.39 ± 1.46^a	0.19 ± 0.09^a	2.62 ± 2.76^a	9.33 ± 3.66^ab	20.40 ± 13.57^b	5.42 ± 2.16^a	23.56 ± 12.54^b
	n			0.56 ± 0.16^bc	0.63 ± 0.19^cd	0.86 ± 0.07^d	0.54 ± 0.13^abc	0.41 ± 0.06^abc	0.32 ± 0.14^ab	0.49 ± 0.09^abc	0.29 ± 0.08^a
Hershey-Bulkley model	G₀	Pa		3.63 ± 5.75^a	3.21 ± 4.08^a	0.06 ± 0.11^a	3.71 ± 5.61^a	10.79 ± 6.97^ab	31.17 ± 20.41^bc	7.94 ± 4.57^a	32.98 ± 16.12^c
	K	Pa s		1.07 ± 1.16^abc	0.46 ± 0.25^ab	0.19 ± 0.09^a	1.28 ± 0.9^abc	4.76 ± 0.65^d	3.07 ± 1.68^cd	2.79 ± 0.95^bcd	5.08 ± 2.31^d
	n			0.63 ± 0.12^ab	0.73 ± 0.09^bc	0.87 ± 0.07^c	0.61 ± 0.1^ab	0.50 ± 0.03^a	0.59 ± 0.14^ab	0.58 ± 0.05^ab	0.48 ± 0.04^a
Viscosity (at 10 s^− 1)			Pa s	0.93 ± 0.89^ab	0.65 ± 0.55^a	0.14 ± 0.05^a	1.58 ± 1.17^ab	3.13 ± 0.87^bc	4.91 ± 2.08^c	2.13 ± 0.77a^b	5.19 ± 1.90^c
Instrinsic viscosity			mgL-1	158.33	148.53	151.56	123.29	293.37	242.31	160.28	147.51

In order to gain an insight into the viscoelastic properties of studied solutions, oscillatory measurements were performed. The G′ is storage modulus, which is a measure of energy stored in material, representing the elastic component of materials stress-strain behavior. The loss modulus G″ is a measure of the energy dissipated through viscous flow, representing the viscous/plastic component of materials stress-strain behavior. All the investigated samples exhibited more solid-like behavior than liquid, as evidenced by storage modulus G′ much higher than loss modulus G″ in amplitude sweep tests. It was confirmed by values of loss factor tanδ < 1 (G’>G’’).

A decrease in the storage modulus G’ was noted as a result of the E1 treatment, for fractions from both sources. Flow point and linear viscoelasticity values slightly decreased for apple, while the for carrot, the opposite trend was observed. The increase in these parameters for carrot suggests that the structure of deacetylated pectins is more resistant to deformation of this material.

A large decrease in the storage modulus G’, observed after removing the arabinose side chains (E2 treatment), combined with an increase in the loss factor G’’, indicates a significant decrease in elastic properties of pectic gels. This may suggest that arabinose chains, as binding points in pectin network, provides a solid-like behavior. For RG-I enriched pectin, arabinose was involved with gel formation under cation and acid conditions and improved network formation and enzymatic debranching resulted in a decrease of side-chain entanglements and hence in looser pectin molecule conformation ²¹. Similarly, the decrease in elastic properties and breaking force was observed for debranched highly methylated citrus pectin gels ²⁷. In this study it was also shown that untreated and debranched pectin gels were governed by the same type of interactions, although for gels being formed by less branched pectins, the network became less entangled with fewer inter-chain connections between the polymer molecules what resulted in overall elasticity decrease. For E2 treatment, increase in the flow point and linear viscoelasticity limit was observed, which proves that the system was able to retain molecular properties of pectin network, as the strain increased. It is worth noting that the E2 variant, by selectively excising the arabinose units, leaves the galactan side chains intact. Therefore, it is possible that aggregate-stabilizing properties of galactans became apparent in this sample, as it was shown by ²⁸. The obtained results of rheological parameters may indicate that for pectins at a concentration of 6%, molecular association occurred with the formation of intermolecular interactions. When E3 enzymes combination was applied, similar viscoelastic parameters for both sources were obtained. This may indicate that degradation of arabinose side chains, deacetylation and removal of rhamnose chains have major effect on the observed differences between DASP pectins of these two materials.

The intrinsic viscosity is a measure of the capacity a polymer molecule to enhance the viscosity of a fluid ²⁹. As a result of enzymatic modifications, only a small effect on this parameter was observed for apple DASP (E3 had the greatest impact on this source). Much more significant effect of enzymatic treatment on intrinsic viscosity was noted in the case of carrot DASP. Enzymatic modifications caused an almost two-fold decrease in this parameter, in particular after E2 and E3 treatments. This shows that the polymers of the DASP fraction with the addition of a buffer form compact molecular aggregates. That may indicate an increase in molecular flexibility and molecules compactness. It can be caused by aggregates dissociation and reduction of intramolecular forces ³⁰, which was induced by selective degradation of the pectin chain.

The different reaction of the apple and carrot fractions to enzymatic modifications may be related to the differences in the monosaccharide composition ¹³. Since solutions were lack of sucrose or cations, apart from the low content of salts in the enzyme buffer, the network formed in the solution is the result of the natural tendency to self-assembly of DASP fraction, which was previously noted and observed on AFM at low concentrations ¹⁰. Therefore, it can be expected that the association of pectin chains of the DASP fraction in aqueous solution in the absence of cations occurs by two mechanisms. For native DASP polymers, composed of both “smooth” and “hairy” structures, neutral side chains are involved in providing multiple short-range attachment points for intermolecular entanglement, what is more favorable than electrostatic repulsion between GalA chains. On the other hand, for “smooth” and linear regions, rich in GalA at neutral pH, strongly charged molecules cause intra-molecular repulsion and thus more extended conformation is formed ⁸. Short linear sections with higher mobility results in interactions between the chains to be more favorable than self-aggregation.

In these studies, changes in the structure and rheological properties of the DASP fraction extracted from apples and carrots, under the influence of enzymes that selectively modify the pectin backbone and side chains were demonstrated. It has been shown, that the structure of pectin in DASP fraction has a significant influence on the rheological properties. This was supported by the changes in the structure, chemical composition and the rheological properties of samples observed as a result of enzymatic modifications of the RG-I fragments. Removal of rhamnose units with simultaneous deacetylation and removal of arabinose side chains resulted in similar rheological parameters of pectic gels from two plant sources, however different from properties of the unprocessed samples. Therefore, it can be concluded that rhamnose may be the factor determining the properties of pectin matrices in solution. Modification with arabinofuranosidase had the greatest impact on the properties of this pectin fraction. Arabinose, present in side chains, is involved in the formation of the pectin network, affecting the pseudoplastic properties and viscosity, but not affecting the mechanical strength in pectin solutions. At the same time, a significant increase in the length of the chains after removal of arabinose indicates that the side chains are hindrance limiting binding of polymer chains.

It can be thus concluded, that the association of pectin chains of the DASP fraction in an aqueous solution in the absence of cations may occur with crucial role of intermolecular interactions, according to two different mechanisms: side chains as short-range attachment points and an extended linear HG conformation favoring inter-chain interactions over self-association.

Pectin source

The research material were apples cv. Najdared (Malus domestica Borkh.) and carrots cv. Brava (Daucus carota subsp. sativus). Material was harvested in Poland in October 2020 and then stored in a cold room at 2°C and normal atmosphere for two days until preparation. Pulp was prepared from 102 kg of raw apples and 34 kg of raw carrots. Both were peeled and sliced. The juice was pressed and the remaining pomace was homogenized. Then the prepared material was frozen at -18°C for further analysis. Alcohol-insoluble residue (AIR) was prepared identically for both plants, according to the method described by ³¹ with some modifications. The pulp was mixed with ~ 70% ethanol (solid–liquid ratio of 1:10, w/v) for 15–30 min, then the mixture was filtered on nylon filter and the residue was stirred again with EtOH. Procedure was repeated until the negative result of the phenol-sulfuric acid test ³², confirming the absence of sugar in the pulp. Next, sample was washed with 96% ethanol and subsequently with acetone, then dried at 45°C.

DASP extraction

Sequential extraction was performed for both sources according to the method proposed by ³³ with certain modifications. AIR was stirred in deionized water (solid:liquid ratio of 1:9, w/v) for 24 h at 21°C and then centrifuged (5 000 rpm). Supernatant was collected as a water-soluble pectins (WSP) fraction and the sediment was mixed with 0.1 M cyclohexane-trans-1.2-diamine tetra-acetate (CDTA, pH 6.5) and stirred at 21°C for 24 h. After centrifugation, the supernatant was separated as a CSP fraction and 0.05 M sodium carbonate (Na₂CO₃), with the addition of 20 mM sodium borohydride (NaBH₄) was added to residue and stirred for 24 h at 21°C. The diluted alkali soluble pectins (DASP) fraction was collected after centrifugation as a supernatant. The DASP fraction was dialyzed in open system using ZelluTrans/Roth® membranes (Carl Roth GmbH & Co. KG, Germany; MWCO 3500 Da) and then crude extract was lyophilized. DASP from apple and carrot was extracted with a yield of 0.44% and 0.61% of fresh weight, respectively.

Determination of degree of acetylation (DA) and degree of methylation (DM)

To determine the degree of methylation (DM) and acetylation (DA) in pectins, samples were saponified with 0.2 M NaOH to produce methanol and acetic acid, which were then measured by HPLC (C18 column, Bionacom velocity LPH-C18, 300 Å, 4.6x250 mm, 5 microns, RI detector). The method of ³⁴ with some modifications by ³⁵ was used. Pectin samples in amount of 5 mg was suspended in 0.5 mL 0.2 M NaOH and incubated in 4 ⁰C for 120 min. Then the mixture was neutralized with 0.2M H₂SO₄ (0.5 mL), centrifuged for 10 min, filtered through 0.22 µm syringe filter and injected onto the HPLC with injection volume 20 µL, mobile phase – 4 mM sulfuric acid at a flow rate: 0.8 mL min^− 1. Standard solutions of methanol and acetic acid were prepared and analyzed at the same conditions. The analysis was performed in triplicate.

Enzymatic treatment

DASP fraction from apples and carrots was treated with enzymes that degrade the RG-I backbone and its side chains. Three types of enzymes were used: RGI acetyl esterase (BtRme NC (CE NC), BT4158, E.C. 3.1.1); rhamnogalacturonan endolyase (BtRge9A (PL9), BT4183, E.C. 4.2.2.23); arabinofuranosidase (CjAbf51B (GH51), E.C. 3.2.1.55). All enzymes were purchased from Nzytech and provided in 35 mM NaHepes buffer, pH 7.5, 750 mM NaCl, 200 mM imidazole, 3.5 mM CaCl₂ and 25% (v/v) glycerol.

The amount of enzymes was selected on the basis of the activity of enzymes ^36–38 and the chemical composition of DASP with 10% excess. Per 1 mg of DASP, 0.22 U of RGL, 9.9 U of RGAE and 2.1 U of ABF were used. According to protein concentrations (RGAE – 0.5 mg mL^− 1; RGL – 0.5 mg mL^− 1; ABF – 0.25 mg mL^− 1) volume values were obtained.

DASP pectins were incubated in three different enzymes cocktails: E1 that was combination of RGAE and RGL; E2 that was ABF only; E3 that was combination of RGAE, RGL and ABF. As a control (no enzymatic treatment), the same buffer solution in which the enzymes were delivered was used.

The pH value of all tested solutions was in the range of 7.30–7.60. Control and enzymes treated samples were incubated in a water bath at 37°C for 120 min. After incubation time, respective samples were cooled in ice bath for 5 min in order to stop enzymatic reaction, mixed and used for further analysis.

AFM imaging and analysis

After enzymatic modifications, 30 µL of each DASP sample in 0.02 mg mL^− 1 concentration was distributed on freshly cleaved mica sheets (EMS, Hatfield, PA, USA) using a POLOS SPIN150i NPP spin coater (SPS-Europe B.V., Putten, the Netherlands). Samples were observed (after drying in a desiccator at 22°C overnight) using a Multimode 8 with a Nanoscope V controller (Bruker, Billerica, MA, USA) with SCANASYST-AIR HR cantilever (Bruker, Billerica, MA, USA) with a nominal spring constant of 0.4 N m^− 1. The observations were conducted in ambient air at a room temperature. The following scan settings were applied: scan size of 4 × 4 µm with resolution equal to 1024x1024 points and scan rate 3.91 Hz. In total, at least 10 images of each sample representing different regions on mica sheets were collected. Preliminary processing of images was carried out using Gwyddion 2.52 ³⁹. Geometrical features of DASP structure were calculated with Matlab R2011a script (MathWorks, Natick, MA, USA). Molecules were characterized using parameters as followed: length of “hairy” molecules, number of segments, segments length and length of “smooth” molecules (Fig. 5).

FT-IR spectroscopy

DASP was dissolved in deionized water with a concentration of 6% (m/v). After preparation samples were vortexed (3 000 rpm) and then mixing overnight. DASP was treated with mixture of E1, E2 or E3 and incubated as described in section 2.4. Subsequently samples were cooled in ice for 5 min and mixing overnight, then freeze-dried. FT-IR spectra of were collected using Nicolet 6700 FT-IR (Thermo Scientific, Madison, WI, USA) with the Smart iTR ATR sampling accessory. All samples from both sources were analyzed under the same conditions. Spectra were collected in the range 4000–650 cm^− 1 with a spectral resolution of 4 cm^− 1. Measurements were performed in three repetitions with 200 scans averaged for each repetition. The baseline corrections were performed using Omnic software (Thermo Scientific). The final average spectrum was calculated from collected data and normalized to 1.0 at 1019 cm^− 1 using Origin Pro 8.5 software (OriginLab Corporation, Northampton, MA, USA).

Determination of rheological properties

DASP 6% (m/v) solutions were prepared in the same way as for FTIR analysis (section 2.6.) and treated with mixture of E1, E2 or E3 and incubated as described in section 2.4. Subsequently samples were cooled in ice for 5 min. For oscillatory test and flow behavior measurements, after cooling, samples were stabilized in room temperature before measurements.

For intrinsic viscosity measurements, a stock solution of 20 mg mL^− 1DASP in deionized water was prepared. A mixture of enzymes (E1, E2 or E3) was added to the well-dissolved sample. After 120 min. if incubation sample was cooled in ice. From the stock solution, dilutions (5–18 mg mL^− 1) were prepared to obtain intrinsic viscosity curves.

Rheological measurements were performed using Discovery Hybrid Rheometer (HR-1) delivered by TA Instruments (New Castle, PA, USA) with a cone plate sensor (40 mm diameter and 2.007° angle) with a 0.56 mm gap between the cone apex and the plate at 20°C.

Viscoelastic properties

Oscillatory test was carried out with amplitude sweeps in order to describe storage (G’) and loss (G’’) modulus of obtained networks. Measurements were performed keeping the frequency at constant value of 0.5 Hz and logarithmic sweep with strain in the range of 0.1 to 50% (25 points per decade, volume of the deposited sample 1 mL).

Flow behaviour

Shear stress vs. shear rate dependences (flow curves) were measured between shear rates of 10–600 s^− 1; 600–10 s^− 1 (logarithmic sweep, 15 points per decade). The viscosity was recorded at constant shear rate of 10 s^− 1. Power law model (Ostwald de Waele’s model) and Herschel–Buckley’s model were applied for obtained flow curves in order to determine the rheological behavior of samples.

Power law model is given as Eq. (1) (Eq. 1):

$$\sigma =K{\gamma }^{n}$$

where $\sigma$ – shear stress; K – consistency index (Pa·sⁿ); γ – shear rate (s^− 1); n – flow behavior index.

Herschel–Buckley’s model is described by the relation (2) (Eq. 2):

$$\sigma ={\sigma }_{0}+K{\gamma }^{n}$$

where σ – shear stress; σ₀– initial shear stress (Pa); K – consistency index (Pa·sⁿ); γ – shear rate (s^− 1); n – flow behavior index.

Intrinsic viscosity

The variable shear rate of 10–400 s^− 1; 400–10 s^− 1 (logarithmic sweep; 25 points per decade) was used to measure viscosity of the solutions with volume of the deposited sample 0.80 mL. Determination of the intrinsic viscosities of samples were obtained via an extrapolation of the belowed function (Eq. 3) to the concentration c equal zero ⁴⁰:

$$\frac{{\eta }_{sp}}{c}=\left[\eta \right]+{k}_{H}{\left[\eta \right]}^{2}c$$

where η_sp is the specific viscosity that can be obtained from the relative viscosity (η _solution/η_solvent); c – polymer solution concentration and k_H is the Huggins constant.

Statistical analysis

The data were analyzed with multi-way ANOVA and post-hoc Tukey HSD test (significance level p < 0.05) using ‘stats’ package (version 4.1.2.), which is a part of R (R Core Team, 2013) and Statistica 13.1 (StatSoft, Cracow, Poland).

Ethics declarations

All methods were in accordance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The botanical material was harvested from a commercial orchard located in Ostrowiec (52° 9′ 59.60″ N, 20° 3’ 23.84′ E) and Agricultural Experimental Station located in Debowa Gora (51° 51,838’ N, 20° 07,176’ E).

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request

Acknowledgements

This work was supported by the National Science Centre, Poland [grant number 2019/35/ O/NZ9/01387]

Author contributions statement

A.K.: Writing – original draft, Conceptualization, Methodology, Formal analysis, Visualization, Investigation. P.M.P: Writing – review &editing, Methodology, Data curation, Conceptualization, Visualization, Validation, Formal analysis. J.C.: Writing – review & editing, Methodology, Investigation, Formal analysis. A.Z.: Writing – review & editing, Resources, Data curation, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript

Competing interests

The authors declare no competing interests.

Voragen, A. G. J., Coenen, G. J., Verhoef, R. P. & Schols, H. A. Pectin, a versatile polysaccharide present in plant cell walls. Struct. Chem. 20, 263–275 (2009).
Zdunek, A., Pieczywek, P. M. & Cybulska, J. The primary, secondary, and structures of higher levels of pectin polysaccharides. Compr. Rev. Food Sci. Food Saf. 20, 1101–1117 (2021).
Haas, K. T., Wightman, R., Peaucelle, A. & Höfte, H. The role of pectin phase separation in plant cell wall assembly and growth. Cell Surf. 7, (2021).
Gawkowska, D., Cieśla, J., Zdunek, A. & Cybulska, J. Cross-linking of diluted alkali-soluble pectin from apple (Malus domestica fruit) in different acid-base conditions. Food Hydrocoll. 92, 285–292 (2019).
Mierczyńska, J., Cybulska, J., Sołowiej, B. & Zdunek, A. Effect of Ca2+, Fe2 + and Mg2 + on rheological properties of new food matrix made of modified cell wall polysaccharides from apple. Carbohydr. Polym. 133, 547–555 (2015).
Brummell, D. A. Cell wall disassembly in ripening fruit. Funct. Plant Biol. 33, 103–119 (2006).
Gilsenan, P. M., Richardson, R. K. & Morris, E. R. Thermally reversible acid-induced gelation of low-methoxy pectin. Carbohydr. Polym. 41, 339–349 (2000).
Gawkowska, D., Cybulska, J. & Zdunek, A. Structure-related gelling of pectins and linking with other natural compounds: A review. Polymers vol. 10 (2018).
Lapasin, R. & Pricl, S. Rheology of Industrial Polysaccharides: Theory and Applications. Rheology of Industrial Polysaccharides: Theory and Applications (1995). doi:10.1007/978-1-4615-2185-3.
Cybulska, J., Zdunek, A. & Kozioł, A. The self-assembled network and physiological degradation of pectins in carrot cell walls. Food Hydrocoll. 43, 41–50 (2015).
Pieczywek, P. M., Kozioł, A., Płaziński, W., Cybulska, J. & Zdunek, A. Resolving the nanostructure of sodium carbonate extracted pectins (DASP) from apple cell walls with atomic force microscopy and molecular dynamics. Food Hydrocoll. 104, 105726 (2020).
Gawkowska, D., Ciesla, J., Zdunek, A. & Cybulska, J. The effect of concentration on the cross-linking and gelling of sodium carbonate-soluble apple pectins. Molecules 24, (2019).
Kaczmarska, A., Pieczywek, P. M., Cybulska, J., Cieśla, J. & Zdunek, A. Structural and rheological properties of diluted alkali soluble pectin from apple and carrot. https://ssrn.com/abstract=4459799.
Zdunek, A., Kozioł, A., Pieczywek, P. M. & Cybulska, J. Evaluation of the Nanostructure of Pectin, Hemicellulose and Cellulose in the Cell Walls of Pears of Different Texture and Firmness. Food Bioprocess Technol. 7, 3525–3535 (2014).
Paniagua, C. et al. Fruit softening and pectin disassembly: An overview of nanostructural pectin modifications assessed by atomic force microscopy. Annals of Botany vol. 114 1375–1383 (2014).
Posé, S., Kirby, A. R., Mercado, J. A., Morris, V. J. & Quesada, M. A. Structural characterization of cell wall pectin fractions in ripe strawberry fruits using AFM. Carbohydr. Polym. 88, 882–890 (2012).
Kacuráková, M., Capek, P., Sasinková, V., Wellner, N. & Ebringerová, A. FT-IR study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses. Carbohydr. Polym. 43, 195–203 (2000).
Chylińska, M., Szymańska-Chargot, M. & Zdunek, A. FT-IR and FT-Raman characterization of non-cellulosic polysaccharides fractions isolated from plant cell wall. Carbohydr. Polym. 154, 48–54 (2016).
Coimbra, M. A., Barros, A., Barros, M., Rutledge, D. N. & Delgadillo, I. Multivariate analysis of uronic acid and neutral sugars in whole pectic samples by FT-IR spectroscopy. Carbohydr. Polym. 37, 241–248 (1998).
Mierczyńska, J., Cybulska, J., Pieczywek, P. M. & Zdunek, A. Effect of Storage on Rheology of Water-Soluble, Chelate-Soluble and Diluted Alkali-Soluble Pectin in Carrot Cell Walls. Food Bioprocess Technol. 8, 171–180 (2015).
Zheng, L. et al. Evaluation of emulsion stability by monitoring the interaction between droplets. LWT 132, (2020).
Chen, J. et al. Pectin Modifications: A Review. Crit. Rev. Food Sci. Nutr. 55, 1684–1698 (2015).
Chen, S. et al. Synergistic gelling mechanism of RG-I rich citrus pectic polysaccharide at different esterification degree in calcium-induced gelation. Food Chem. 350, 129177 (2021).
Pieczywek, P. M., Cieśla, J., Płaziński, W. & Zdunek, A. Aggregation and weak gel formation by pectic polysaccharide homogalacturonan. Carbohydr. Polym. 256, (2021).
Sims, I. M., Smith, A. M., Morris, G. A., Ghori, M. U. & Carnachan, S. M. Structural and rheological studies of a polysaccharide mucilage from lacebark leaves (Hoheria populnea A. Cunn.). Int. J. Biol. Macromol. 111, 839–847 (2018).
Alba, K., Kasapis, S. & Kontogiorgos, V. Influence of pH on mechanical relaxations in high solids LM-pectin preparations. Carbohydr. Polym. 127, 182–188 (2015).
Sousa, A. G., Nielsen, H. L., Armagan, I., Larsen, J. & Sørensen, S. O. The impact of rhamnogalacturonan-I side chain monosaccharides on the rheological properties of citrus pectin. Food Hydrocoll. 47, 130–139 (2015).
Makshakova, O. N., Faizullin, D. A., Mikshina, P. V., Gorshkova, T. A. & Zuev, Y. F. Spatial structures of rhamnogalacturonan I in gel and colloidal solution identified by 1D and 2D-FTIR spectroscopy. Carbohydr. Polym. 192, 231–239 (2018).
Torralbo, D. F., Batista, K. A., Di-Medeiros, M. C. B. & Fernandes, K. F. Extraction and partial characterization of Solanum lycocarpum pectin. Food Hydrocoll. 27, 378–383 (2012).
Kontogiorgos, V., Margelou, I., Georgiadis, N. & Ritzoulis, C. Rheological characterization of okra pectins. Food Hydrocoll. 29, 356–362 (2012).
Renard, C. M. G. C. Variability in cell wall preparations: Quantification and comparison of common methods. Carbohydr. Polym. 60, 515–522 (2005).
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 28, 350–356 (1956).
Redgwell, R. J. & Selvendran, R. R. Structural features of cell-wall polysaccharides of onion Allium cepa. Carbohydr. Res. 157, 183–199 (1986).
Levigne, S., Thomas, M., Ralet, M. C., Quemener, B. & Thibault, J. F. Determination of the degrees of methylation and acetylation of pectins using a C18 column and internal standards. Food Hydrocoll. 16, 547–550 (2002).
Yu, Y. et al. Comparison of analytical methods for determining methylesterification and acetylation of pectin. Appl. Sci. 11, (2021).
Beylot, M.-H., Mckie, V. A., Voragen, A. G. J., Doeswijk-Voragen, C. H. L. & Gilbert, H. J. The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity. Biochem. J vol. 358 (2001).
Searle-van Leeuwen, M. J. F., van den Broek, L. A. M., Schols, H. A., Beldman, G. & Voragen, A. G. J. Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins. Appl. Microbiol. Biotechnol. 38, 347–349 (1992).
Silva, I. R., Jers, C., Meyer, A. S. & Mikkelsen, J. D. Rhamnogalacturonan I modifying enzymes: An update. New Biotechnology vol. 33 41–54 (2016).
Nečas, D. & Klapetek, P. Gwyddion: An open-source software for SPM data analysis. Central European Journal of Physics vol. 10 181–188 (2012).
Huggins, M. L. The Viscosity of Dilute Solutions of Long-Chain Molecules. IV. Dependence on Concentration. J. Am. Chem. Soc. 64, 2716–2718 (1942).

No competing interests reported.

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Effect of enzymatic modification on the structure and rheological properties of diluted alkali soluble pectin fraction rich in RG-I

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Abstract

Figures

Introduction

Results and discussion

Nanostructure

Functional groups

Rheological properties

Conclusions

Materials and Methods

Pectin source

DASP extraction

Determination of degree of acetylation (DA) and degree of methylation (DM)

Enzymatic treatment

AFM imaging and analysis

FT-IR spectroscopy

Determination of rheological properties

Viscoelastic properties

Flow behaviour

Intrinsic viscosity

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1